Removal Of Leucine Research Articles

Prolonged deprivation of arginine or leucine induces PI3K/Akt-dependent reactivation of mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase complex that promotes anabolic processes including protein, lipid, and nucleotide synthesis, while suppressing catabolic processes such as macroautophagy. mTORC1 activity is regulated by growth factors and amino acids, which signal through distinct but integrated molecular pathways: growth factors largely signal through the PI3K/Akt-dependent pathway, whereas the availabilities of amino acids leucine and arginine are communicated to mTORC1 by the Rag-GTPase pathway. While it is relatively well described how acute changes in leucine and arginine levels affect mTORC1 signaling, the effects of prolonged amino acid deprivation remain less well understood. Here, we demonstrate that prolonged deprivation of arginine and/or leucine leads to reactivation of mTORC1 activity, which reaches activation levels similar to those observed in nutrient-rich conditions. Surprisingly, we find that this reactivation is independent of the regeneration of amino acids by canonical autophagy or proteasomal degradation but is dependent on PI3K/Akt signaling. Together, our data identify a novel crosstalk between the amino acid and PI3K/Akt signaling pathways upstream of mTORC1. These observations extend our understanding of the role of mTORC1 in growth-related diseases and indicate that dietary intervention by removal of leucine and/or arginine may be an ineffective therapeutic approach.

Chronic leucine exposure results in reduced but reversible glucose-stimulated insulin secretion in INS-1 cells

Previous studies have demonstrated that sustained high leucine exposure decreases glucose-stimulated insulin secretion (GSIS). However, whether this effect is recoverable following the removal of leucine is unclear. Pancreatic/duodenal homeobox-1 (PDX-1) and its downstream target, glucose transporter 2 (GLUT2), are reported to be positively associated with insulin secretion. However, it also remains unclear whether the effect of leucine on GSIS is accompanied by alterations in PDX-1 and GLUT2. In the present study, insulin secretion, insulin content, PDX-1 and GLUT2 protein expression in INS-1 (rat insulinoma cell line) cells were assessed following a 24-h incubation in 40 mmol/l leucine. Half of the cells were incubated in leucine-free media for a further 24 h to observe the abovementioned effects. In contrast to the control, 40 mmol/l leucine for 24 or 48 h diminished GSIS at high glucose concentrations by 11% (P=0.026) or 22% (P=0.003), insulin content by 14% (P=0.008) or 20% (P=0.002), as well as decreasing PDX-1 and GLUT2 expression. When leucine was removed from the media for a further 24-h incubation, in comparison with those cells that were maintained in leucine treatment for 24 and 48 h, the high GSIS increased by 13% (P=0.032) and 27% (P=0.002), insulin content was augmented by 10% (P=0.014) and 20% (P=0.003), and the protein expression of PDX-1 and GLUT2 also increased. The present study demonstrates that sustained high concentrations of leucine induce a reversible impairment of GSIS and alter insulin content, which is mediated by PDX-1 and GLUT2, in INS-1 cells.

Secreted 3-Isopropylmalate Methyl Ester Signals Invasive Growth during Amino Acid Starvation in Saccharomyces cerevisiae

The Saccharomyces cerevisiae methyltransferase encoded by TMT1 catalyzes the AdoMet-dependent monomethylation of 3-isopropylmalate, an intermediate of the leucine biosynthetic pathway. The biological significance of methylating 3-isopropylmalate and the relationship between Tmt1 and the leucine biosynthetic pathway is not yet established. We present evidence here showing that methylation of 3-isopropylmalate functions to extracellularly signal yeast to grow invasively. We show that methyl esterification generates 3-isopropylmalate-1-methyl ester. We find that the Tmt1 methyltransferase functions independently of the biosynthetic pathway but is induced when cells are starved for amino acids; the largest induction is observed with the removal of leucine from the media. This amino acid starvation stress response is controlled by the transcriptional activator Gcn4. After methylation, 3-isopropylmalate methyl ester is secreted into the media within 3 h. Thin layer chromatography and gas chromatography mass spectroscopy confirm that the intact molecule is secreted. Finally, we show that purified 3-isopropylmalate methyl ester can enhance the ability of the haploid yeast strain 10560-23C to grow invasively. Our data identifies 3-isopropylmalate methyl ester as an autoinductive molecule that provides a signal to yeast to switch from vegetative to invasive growth in response to amino acid starvation.

Rheb Binding to Mammalian Target of Rapamycin (mTOR) Is Regulated by Amino Acid Sufficiency

The removal of extracellular amino acids or leucine alone inhibits the ability of the mammalian target of rapamycin (mTOR) to signal to the raptor-dependent substrates, p70 S6 kinase and 4E-BP. This inhibition can be overcome by overexpression of the Rheb GTPase. Rheb binds directly to the amino-terminal lobe of the mTOR catalytic domain, and activates mTOR kinase in a GTP-dependent manner. Herein we show that the binding of Rheb to endogenous and recombinant mTOR is reversibly inhibited by withdrawal of all extracellular amino acids or just leucine. The effect of amino acid withdrawal is not attributable to changes in Rheb-GTP charging; amino acid withdrawal does not alter the GTP charging of recombinant Rheb. Moreover, the binding of mTOR to Rheb mutants that are unable to bind guanyl nucleotide in vivo is also inhibited by amino withdrawal. The inhibitory effect of amino acid withdrawal is exerted through an action on mTOR, at a site largely distinct from that responsible for the binding of Rheb; deletion of the larger, carboxyl-terminal lobe of the mTOR catalytic domain eliminates the inhibitory effect of amino acid withdrawal on Rheb binding, without altering Rheb binding per se. The lesser ability of the mTOR catalytic domain to bind Rheb after amino acid withdrawal does not persist after extraction and purification of the mTOR polypeptide. Amino acid withdrawal may generate an inhibitor of the Rheb-mTOR interaction that interferes with the signaling function of TOR complex 1.

Use of a deletion approach to assess the amino acid requirements for optimum fermentation by mixed micro-organisms from the sheep rumen

AbstractAmino acids stimulate the growth rate and growth yield of ruminal micro-organisms, but the basis of this stimulation, in terms of amino acids which most limit growth, has never been fully established. Here, for the first time, a deletion approach was investigated using in vitro incubations of mixed ruminal micro-organisms supplied with a mixture of xylose, starch and cellobiose as energy sources and ammonia plus a complete amino acids mixture or mixtures with a single amino acid omitted as nitrogen sources, enabling the evaluation of the impact on ruminal fermentation of the deletion of a single amino acid from a complete amino acids mixture. Significant effects (P < 0·05) on total gas production were observed after 10 h of incubation when glutamate, glutamine, isoleucine, leucine, phenylalanine, serine, tryptophan or tyrosine were deleted from the amino acids mixture. The only significant effect of an amino acid deletion on volatile fatty acid production at 10 h was with serine (P < 0·05), although the effect of omitting others, including arginine, isoleucine, leucine and phenylalanine, approached significance (P < 0·01). The removal of leucine caused a 0·09 decrease in growth yield (P < 0·05); no other deletion affected the yield significantly (P > 0·05). Net gas production for each treatment was calculated by subtracting gas production in the absence of carbohydrates from gas production in their presence, thus eliminating gas production from amino acids from the values. At all times up to 10 h, the most significant effects on net gas production were found when serine, leucine, or the aromatic amino acids were omitted from the amino acids mixture. Thus, the deletion approach confirmed that no single amino acid limits ruminal fermentation more than any other, although a few, principally phenylalanine, leucine and serine, have a particularly significant rôle in the ruminal fermentation rate of soluble, rapidly degraded materials and/or microbial growth efficiency.

Diagnosis and treatment of maple syrup disease: a study of 36 patients.

To evaluate an approach to the diagnosis and treatment of maple syrup disease (MSD). Family histories and molecular testing for the Y393N mutation of the E1alpha subunit of the branched-chain alpha-ketoacid dehydrogenase allow us to identify infants who were at high risk for MSD. Amino acid concentrations were measured in blood specimens from these at-risk infants between 12 and 24 hours of age. An additional 18 infants with MSD were diagnosed between 4 and 16 days of age because of metabolic illness. A treatment protocol for MSD was designed to 1) inhibit endogenous protein catabolism, 2) sustain protein synthesis, 3) prevent deficiencies of essential amino acids, and 4) maintain normal serum osmolarity. Our protocol emphasizes the enhancement of protein anabolism and dietary correction of imbalances in plasma amino acids rather than removal of leucine by dialysis or hemofiltration. During acute illnesses, the rate of decrease of the plasma leucine level was monitored as an index of net protein synthesis. The treatment protocol for acute illnesses included the use of mannitol, furosemide, and hypertonic saline to maintain or reestablish normal serum sodium and extracellular osmolarity and thereby prevent or reverse life-threatening cerebral edema. Similar principles were followed for both sick and well outpatient management, especially during the first year, when careful matching of branched-chain amino acid intake with rapidly changing growth rates was necessary. Branched-chain ketoacid excretion was monitored frequently at home and branched-chain amino acid levels were measured within the time of a routine clinic visit, allowing immediate diagnosis and treatment of metabolic derangements. 1) Eighteen neonates with MSD were identified in the high-risk group (n = 39) between 12 and 24 hours of age using amino acid analysis of plasma or whole blood collected on filter paper. The molar ratio of leucine to alanine in plasma ranged from 1.3 to 12.4, compared with a control range of 0.12 to 0.53. None of the infants identified before 3 days of age and managed by our treatment protocol became ill during the neonatal period, and 16 of the 18 were managed without hospitalization. 2) Using our treatment protocol, 18 additional infants who were biochemically intoxicated at the time of diagnosis recovered rapidly. In all infants, plasma leucine levels decreased to <400 micromol/L between 2 to 4 days after diagnosis. Rates of decrease of the plasma leucine level using a combination of enteral and parenteral nutrition were consistently higher than those reported for dialysis or hemoperfusion. Prevention of acute isoleucine, valine, and other plasma amino acid deficiencies by appropriate supplements allowed a sustained decrease of plasma leucine levels to the therapeutic range of 100 to 300 micromol/L, at which point dietary leucine was introduced. 3) Follow-up of the 36 infants over >219 patient years showed that, although common infections frequently cause loss of metabolic control, the overall rate of hospitalization after the neonatal period was only 0.56 days per patient per year of follow-up, and developmental outcomes were uniformly good. Four patients developed life-threatening cerebral edema as a consequence of metabolic intoxication induced by infection, but all recovered. These 4 patients each showed evidence that acutely decreased serum sodium concentration and decreased serum osmolarity were associated with rapid progression of cerebral edema during their acute illnesses. Classical MSD can be managed to allow a benign neonatal course, normal growth and development, and low hospitalization rates. However, neurologic function may deteriorate rapidly at any age because of metabolic intoxication provoked by common infections and injuries. Effective management of the complex pathophysiology of this biochemical disorder requires integrated management of general medical care and nutrition, as well as control of several variables that influence endogenous protein anabolism and catabolism, plasma amino acid concentrations, and serum osmolarity.

A new concept for the mechanism of action of chymotrypsin: the role of the low-barrier hydrogen bond.

The basicities of the diad H57-D102 at N(epsilon)2 in the tetrahedral complexes of chymotrypsin with the peptidyl trifluoromethyl ketones (TFK) N-acetyl-L-Leu-DL-Phe-CF3 and N-acetyl-DL-Phe-CF3 have been studied by 1H-NMR. The protons bridging His 57 and Asp 102 in these complexes are engaged in low-barrier hydrogen bonds (LBHBs). In 1H-NMR spectra at pH 7.0, these protons appear at delta 18.9 and 18.6 ppm, and the pK(a)s of the diads are 12.0 +/- 0.2 and 10.8 +/- 0.1, respectively. The difference indicates that removal of leucine from the second aminoacyl site S2 of chymotrypsin weakens the LBHB and decreases the basicity of the H57-D102 diad relative to the case in which S2 is occupied by leucine. Consideration of the available structural data on chymotrypsin and other serine proteases, together with the high pK(a)s of the hemiketals formed with TFKs, suggests that LBHB formation in catalysis arises through a substrate-induced conformational transition leading to steric compression between His 57 and Asp 102. Because the N-O distance in the LBHB is shorter than the Van der Waals contact distance, the LBHB is proposed to stabilize the tetrahedral intermediate through relief of steric strain between these residues. In this mechanism, substrate-induced steric compression within the diad increases the basicity of N(epsilon)2 in His 57, making it a more effective base for abstracting a proton from Ser 195 in the formation of the tetrahedral intermediate. The values of pK(a) for N(epsilon)2 in TFK adducts lie between those of Ser 195 (pK(a) approximately 14) and the leaving group in tetrahedral adducts (pK(a) approximately 9), making N(epsilon)2 of the H57-D102 diad strong enough as a base to abstract the proton from Ser 195 in tetrahedral adduct formation but not so strong that its conjugate acid cannot protonate the leaving group. According to this theory, the "normal" pK(a) of His 57 in free chymotrypsin arises from the use of part of the stabilization energy provided by the LBHB to drive the conformational compression required for its formation. In catalysis, the energy for conformational compression is supplied by the binding of remote portions of the substrate, including the side chains of P1 and P2.

Physiological concentrations of L-leucine control the release of prolactin from cultured rat pituitary cells.

In an attempt to further define the optimum experimental conditions for the in vitro study of PRL endocrinology, the effect of extracellular leucine concentration on PRL secretion was assessed. Rat pituitary cells, maintained in short term monolayer culture, were washed free of serum and placed in Minimum Essential Medium (MEM; 0.39 mM leucine) supplemented with nonessential amino acids and 0.1% gelatin. After the establishment of a constant rate of basal PRL release, the MEM was replaced with leucine-free MEM (supplemented as above), and the rate of PRL release was followed. PRL release from the cells in the leucine-free medium fell within 15 min to a rate 30% of that seen in the controls. Return to the control rate was achieved by the addition of L-leucine only. The D-isomer, isoleucine, valine, proline, leucyl-leucine, and leucine-agarose were ineffective. In the short term, the rate of PRL synthesis was found to be independent of extracellular leucine and the inhibition of release seen upon removal of leucine, and the subsequent recovery upon replacement of leucine was also observed when this experiment was conducted in the presence of 50 micrograms/ml cycloheximide. The L-leucine recovery was found to be dose dependent, with an increase in leucine concentration from 20 to 40 mg/liter causing a tripling of the PRL release rate. The addition of 10(-7) M TRH showed that leucine can be limiting in the response to this stimulant and allowed a response to as little as 10 mg/liter leucine to be discerned. From these experiments, it is concluded that physiological concentrations of leucine specifically and profoundly affect the release of PRL by a mechanism independent of protein synthesis. This leucine requirement for release has not been previously reported.

Repression of Escherichia coli pyridine nucleotide transhydrogenase by leucine

Addition of 0.1% casein hydrolysate to a minimal growth medium decreased membrane-bound transhydrogenase activity in Escherichia coli by about 80%. Of the amino acids added individually to the growth medium, only leucine and, to a lesser extent, methionine and alanine were effective, alpha-Ketoisocaproate- and leucine-containing peptides repressed the activity, and leucine also repressed activity in adenyl cyclase-deficient and relaxed strains. Derepression of transhydrogenase followed the removal of leucine from the growth medium and was sensitive to rifampin and chloramphenicol. A phosphoglucoisomerase-deficient strain that was forced to use the hexose monophosphate shunt exclusively had normal levels of transhydrogenase, which was repressed by leucine. Transhydrogenase activity doubled in mutants lacking either of the shunt dehydrogenases but was still repressed by leucine. In strains constitutive for the leucine biosynthetic operon, transhydrogenase was repressed by leucine but in strains livR and lst R, with leucine transport resistant to leucine repression, transhydrogenase was not repressed by leucine. These data suggest that transhydrogenase may have a function in the transport of branched-chain amino acids. In a hisT strain (which has altered leucyl-tRNA), transhydrogeanse was at a repressed level without the addition of leucine, suggesting that leucyl-tRNA may be involved in the regulation.

Effect of inhibition of protein synthesis on lipid metabolism in Lactobacillus plantarum.

In Lactobacillus plantarum 17-5, lipid synthesis appears to be correlated with protein synthesis. Inhibition of protein synthesis by chloramphenicol (50 mug/ml) caused the nearly simultaneous inhibition of incorporation of radioactive oleic acid into polar lipids before the cessation of growth. In addition, de novo fatty acid synthesis, as determined by the incorporation of radioactive acetate into cellular lipids, was also inhibited. Removal of the antibiotic resulted in the resumption of growth, protein synthesis, and polar lipid synthesis. Inhibition of protein synthesis by leucine deprivation also produced a marked reduction in the incorporation of radioactive oleic acid into the total polar lipids at about the same time that growth stopped (30 to 60 min after the removal of leucine). However, the different classes of lipids behaved differently. For example, the incorporation of oleic acid into cardiolipin was inhibited immediately upon removal of leucine from the cultures, whereas incorporation into phosphatidyl-glycerol was maintained at near normal rates for 60 min after the removal of leucine and then ceased. In contrast, the accumulation of radioactive oleic acid in a neutral lipid identified as diglyceride occurred to a much greater extent in leucine-deprived cultures than in control (+ leucine) cultures. Upon addition of leucine to leucine-deprived cultures, the rates of synthesis of phosphatidyl-glycerol and cardiolipin returned to normal; the amount of radioactivity in the diglyceride fraction decreased to normal levels concomitantly with increased phospholipid synthesis.

Efficiency of α-Ketoisocaproic Acid as a Substitute for Leucine in the Diet of the Growing Rat

Immature male albino rats (70-80 g), eating a diet furnishing all nutrients for optimal growth and containing 84.6 mumoles of leucine/g, gained weight at an average rate of 5.9 g/day. Removal of leucine caused an average daily weight loss of 1.3 g. Addition of leucine to the diet in graduated amounts corrected this weight loss, the improvement in body weight (BW) being proportional to the dietary content of leucine. Addition of alpha-ketoisocaproic acid in place of leucine to the leucine-free diet also prevented loss of BW. Percentage efficiency of alpha-ketoisocaproic acid as a dietary substitute for leucine was calculated as: (see article). Efficiency of such substitution varied from 27% for a diet containing 84.6 mumoles of alpha-ketoisocaproic acid/g to 20% for a diet containing 169.2 alpha-ketoisocaproic acid/g.

Effect of hemorrhagic shock, fasting, and corticosterone administration on leucine oxidation and incorporation into protein by skeletal muscle

The metabolism of leucine by rabbit skeletal muscle was studied 1 and 3 days after shock or glucocorticoid administration. Hemorrhagic shock resulted in an increase in the capacity for leucine oxidation and a concomitant suppression of leucine incorporation into protein by skeletal muscle. This defect persisted, in part, for at least 3 days following injury. Corticosterone administration to normal animals produced an increase in leucine oxidation and a decrease in leucine incorporation into protein similar to that observed following shock. Fasting, in contrast, caused a simultaneous decrease in both leucine oxidation and incorporation into protein. These findings are consistent with the possibility that the net protein catabolism associated with hemorrhagic shock may be promoted by removal of leucine from the cellular protein synthetic amino acid pool through irreversible combustion. Shock-induced adrenal steroid secretion may contribute to the observed changes in leucine metabolism by skeletal muscle.

Reversible arrest of Chinese hamster cells in G1 by partial deprivation of leucine

The cell cycle of Chinese hamster cells in culture can be arrested by removal of leucine from the medium. When a growing culture is completely deprived of leucine, part of the cell population accumulates in G1, but some cells are arrested in the S and G2 periods. When a trace of leucine (0.1% or 1.0% of the normal concentration) is left in the medium, more of the cells accumulate in G1 during a 36-h period of deprivation. The maximum G1 accumulation has been obtained using 0.1% of the normal leucine concentration with a culture of low cell density. Release of the culture by addition of leucine is followed by a moderate amount of synchrony in DNA synthesis and cell division. The experiments show that cells can complete the S, G2, and division periods with levels of leucine in the medium that do not permit cells to traverse through G1. This differential sensitivity of G1 appears to be positioned several hours before the initiation of DNA synthesis. In addition, the progress of cells in middle and late G1 at the time of leucine withdrawal is cancelled, and such cells revert to an earlier position in G1 where they remain in cycle arrest unless released by reprovision of leucine.